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Need help!!molecular cloning

Need help!!molecular cloning - DNA Techniques

Need help!!molecular cloning - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #1  
Old 08-29-2007, 02:03 PM
Pipette Filler
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Unhappy Need help!!molecular cloning



Hello everybody,
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?
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Old 09-12-2007, 12:43 PM
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Default Re: Need help!!molecular cloning

Hello Bsengez,

you may have:

1) analyzed your restriction sites wrong
2) added an extra restriction enzyme by accident
3) cloned in a restriction site (present in the insert!) - this is a big problem

I would analyze your plasmid and your insert with webcutter.

Cheers
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Old 10-09-2007, 09:00 PM
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Default Re: Need help!!molecular cloning

The restriction sites are okey.
I have cloned it in right positions.
I analyzed my vector in webcutter, thanks for all your attention.
I believed that I haven't added a wrong enzyme, but you know everyone can make mistakes.
Now, I overcome my problems. When I have enough time, I will check it again.
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Old 11-08-2007, 11:34 AM
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Default Re: Need help!!molecular cloning

Hi there,
I believe it is either a partial digest or star activity. Which enzyme are you using for your test digestions?. Cheers!
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Old 11-12-2007, 08:44 PM
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Default Re: Need help!!molecular cloning

I'm using XhoI, NotI, HindIII, XbaI. There is no star activity with the enzymes and the buffer I used. Thanks, but I have already thought about this possibility.
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